Field of Invention: The present invention is a new method for monitoring water for microbiota. Specific organisms are identified optically and electronically in a continuous, real-time process usmg specific, customized software and commercially available equipment. The method will archive analog and digital images of the organisms. The type and frequency of the identified water-borne organisms will be immediately employed to alter wastewater treatment processes or to close drinking water intake pipes.
Description of Prior Art: Other technology that can use continuous, real-time analysis is of two basic types. One method is based on the size of the organism (particle counters), and the other is based on fluorescence of the organism (flow cytometers). Particle counters have serious limitations because they can only distinguish a narrow range of sizes and they cannot distinguish the type of organisms within the size range (Sprules et al., 1992). Also there is no way to distinguish inanimate particles from living organisms. A commercially available counter is the Optical Plankton Counter Model OPC-LT, OPC-IL, OPC-2D by Focal Technologies, Inc. Dartmouth, Nova Scotia.
The flow cytometry machines utilize fluorescence to identify the organisms either through autofluorescence the wavelength of which would be specific for the organism or through specific fluorochrome dyes linked to antibodies that are organism specific (Brailsford and Gatley, 1993). With this system specific identification can be made, but only if the organism autofluoresces at its specific wavelength. If it does not autofluoresce, fluorochrome-antibody complexes have to be made for a specific organism. Thus the flow cytometry system is very limited: limited to the organisms that emit specific wavelengths, if they autofluoresce, and limited by the necessity to make specific antibodies to a very large variety and range of organisms to anticipate the available organisms. Thus the increase in specificity comes with a decrease in speed, over the particle counter. Further, the machine is extremely expensive, approximately 10 times greater than our method. It is also much more expensive to maintain and operate. A commercially available machine is made by Coulter, model EPICS 741.
To overcome some of the drawbacks, a combination of the two technologies (looking at fluorescence of the particles) has been tried (Porter et al., 1995). Thus there is an increase in specificity and decrease in speed over the standard particle counter. A commercially available machine is the Becton Dickenson FACStar Plus. But the immunofluorescent flow cytometry/ particle counter system is still a particle counter that makes identification indirectly, that requires great expertise and cost to operate, and will be difficult to operate in real time.
The current invention, the Continuous Microbiotal Recognition Method is superior to the above technology. High specificity is obtained with phase microscope images coupled with software to identify specific organisms makes this technology relatively fast speed and relatively low cost (Das et al., 1996).
References Cited
Brailsford, M. and S. Gatley 1993. Rapid analysis of microorganisms using flow cytometry. in: Lloyd, D. ed. 1993. Flow Cytometry in Microbiology. Springer Verlag, London.
Das, M, F. Butterworth, and R. Das. 1996. Statistical signal modeling techniques for automated recognition of water-borne microbial shapes. Proc. 39th Midwest Symposium on Circuits and Systems pp. 613-616. IEEE Press, Piscataway.
Porter, J., , J. Robinson, C. Edwards, 1995 Recovery of a bacterial sub-population from sewage using immunofluorescent flow cytometry and cell sorting. FEMS microbiology letters. 133 (n 1/2):195.
Sprules, W. G., B. Bergstrom, H. Cyr, B. R. Hargreaves, S. S. Kilham, H. J. MacIsaac, K. Matsushita, R. S. Stemberger, R. Williams. 1992. Non-video optical instruments for studying zooplankton distribution and abundance. Arch. Hydrobiol. Beih., 36:45"58.
BRIEF SUMMARY OF THE INVENTION
The method is an automated, continuous, opto-electronic system designed to recognize and identify microbiota in water with the purpose of assessing (1) the quality and extent of sewage digestion, (2) the presence of pathogens in drinking water, and (3) the biological status of natural waters. It involves two separate stages: sampling/distribution and imaging/processing/identification. First, the water to be tested is pumped into the sampling/distribution part of the Continuous Microbiotal Recognition system where aliquots are moved into a viewing chamber of a microscope. In the second stage, microbiota will be imaged by video, the images will be captured digitally, and a CPU with peripheral and I/O devices will process, archive and act on this captured information. The images will be processed where decisions based on a fuzzy decision tree in the CPU can be made to issue warnings to personnel and/or commands to close or open valves, transmitted via modem and phone lines/local area networks, by an actuator/annunciator device.
Thus the Continuous Microbiotal Recognition Method is superior to the particle counting, flow cytometry or combined methods because it is able to identify specific organisms, over a wider range of sizes and categories, in real time with far greater certainty, at far less cost.